Tropane alkaloids, such as hyoscyamine, 6β-hydroxyhyoscyamine and scopolamine, are secondary metabolites that were traditionally applied in medicine due to their anticholinergic activity. Hyoscyamine is converted into 6β-hydroxyhyoscyamine and scopolamine by Hyoscyamine-6β-hydroxylase (H6H). Nowadays, these bioactive compounds are obtained from natural producer plants due to the cost and complexity of their chemical synthesis. In the present work we explored the development of an alternative strategy for the production of the most valuable alkaloids, 6β-hydroxyhyoscyamine and scopolamine, using Escherichia coli harboring the H6H enzyme as biocatalysts. In addition, the protein extracts of the induced bacteria were assayed for the transformation of hyoscyamine into the more valuable alkaloids. For this purpose the h6hcDNA, previously amplified from Brugmansia candida total RNA preparations, was inserted in frame to the trx tag into the pET32a(+) vector. E. coli Origami strains were used as host for the expression. The strategy allowed us to produce enough quantities of a soluble and functional enzyme. Protein extracts and whole cells of the induced bacteria were able to transform hyoscyamine into the valuable products. In addition, we found that except from 2-oxoglutarate, no supplementation of the reaction mixture with the cofactors and co-substrates was needed. The process developed in this work is attractive since it could become an alternative to the traditional isolation of 6β-hydroxyhyoscyamine and scopolamine.